The SPIM (single plane illumination microscopy) technique, in which a sample is illuminated in layers, allows a quicker detection, which is also gentler on the sample, of image data compared to a scanning of a sample at specific points, for example. A known field of use of SPIM technology is the field of fluorescence microscopy, wherein fluorophores are excited in the sample by means of laser light. In the case of SPIM technology, an excitation takes place here only in one plane by an illumination light sheet. Damage to the sample caused by illumination light in other planes is hereby avoided.
An optical device that functions in accordance with the SPIM method is described in DE 102 57 423 A1. In the case of this microscope, a sample is illuminated with a thin light sheet, whilst being observed perpendicularly to the plane of the illuminating light sheet. Here, the illumination and the detection take place over two separate optical beam paths each with a separate optics, in particular with two separate objectives perpendicular to one another.
DE 10 2009 044 983 A1 discloses a microscope which comprises an illumination means with which a light sheet for illuminating a sample region is generated, which light sheet is extended approximately flat in the direction of an illumination axis of an illumination beam path and in the direction of a transverse axis disposed transverse to the illumination axis. The microscope additionally comprises a detection means with which light irradiated from the sample region along a detection axis of a detection beam path is detected, wherein the illumination axis and detection axis and also the transverse axis and detection axis are arranged at an angle with respect to one another different from zero, and wherein the detection means additionally comprises a detection objective in the detection beam path. In the case of a microscope of this kind, the detection means additionally comprises an optical detection element, which is arranged spatially separate from a front lens of the detection objective, is adjustable independently thereof, and by means of which the size of a detection image field can be continuously varied, and/or by means of which a detection focus plane in the sample region can be continuously adjusted.
Dean, K. & Fiolka, R., “Uniform and scalable light-sheets generated by extended focusing”, Opt. Express 22, 26141-26152 (2014) discloses a system in which a thin, short illumination beam with a quickly tunable lens is moved back and forth along the optical axis. Due to the shallower depth of field of the beam, said beam can be thinner, which should increase the image contrast. However, since fluorescence is excited also in the out-of-focus region of the beam, a slit diaphragm is used in order to achieve the desired effect. This slit diaphragm runs in a conjugated plane in the detection beam path in a collinear manner with the illumination beam. In spite of the use of the slit diaphragm, the increase of the image contrast is relatively small, in particular because a lot of fluorescence light is detected that has not been excited by the focus of the illumination beam.
Zong, W. et al. “Large-field high-resolution two-photon digital scanned light-sheet microscopy” Cell Res. (2014) discloses a similar microscope, wherein, however, a non-linear fluorescence excitation is provided there. A microscope of this kind is also described in CN 104407436.
In Gao, L., “Extend the field of view of selective plan illumination microscopy by tiling the excitation light sheet”, Opt. Express, 23, 6102-6111 (2015), it is disclosed to displace the light sheet generated by a scanned illumination beam in a plurality of separate steps along the illumination axis and to then join together the images obtained individually for each step. This method has the disadvantage that it takes a long time to record a number of individual images one after the other. Furthermore, the effective thickness of the illuminated region is not uniform over the field of view along the direction of propagation of the illuminating beam. The illuminating light beam is always thinner in the middle of the individual images than at the edge. The image formed from individual images thus consists in alternation of regions which have been illuminated by thinner and thicker light beams, that is to say with higher and lower contrast.